Inhibitors of Akt activity

ABSTRACT

The present invention is directed to compounds comprising a triazolo[4,3-b]pyridazine moiety which inhibit the activity of Akt, a serine/threonine protein kinase. The invention is further directed to chemotherapeutic compositions containing the compounds of this invention and methods for treating cancer comprising administration of the compounds of the invention.

This application is a 371 of PCT/GB02/01649, filed Apr. 8, 2002, whichclaims priority under Title 35, United States Code 119(e) fromProvisional Application Ser. No. 60/282,806, filed Apr. 10, 2001.

BACKGROUND OF THE INVENTION

The present invention relates to triazolo[4,3-b]pyridazine containingcompounds that are inhibitors of the activity of one or more of theisoforms of the serine/threonine kinase, Akt (also known as PKB). Thepresent invention also relates to pharmaceutical compositions comprisingsuch compounds and methods of using the instant compounds in thetreatment of cancer.

Apoptosis (programmed cell death) plays essential roles in embryonicdevelopment and pathogenesis of various diseases, such as degenerativeneuronal diseases, cardiovascular diseases and cancer. Recent work hasled to the identification of various pro- and anti-apoptotic geneproducts that are involved in the regulation or execution of programmedcell death. Expression of anti-apoptotic genes, such as Bcl2 orBcl-x_(L), inhibits apoptotic cell death induced by various stimuli. Onthe other hand, expression of pro-apoptotic genes, such as Bax or Bad,leads to programmed cell death (Aams et al. Science, 281:1322-1326(1998)). The execution of programmed cell death is mediated by caspase-1related proteinases, including caspase-3, caspase-7, caspase-8 andcaspase-9 etc (Thornberry et al. Science, 281:1312-1316 (1998)).

The phosphatidylinositol 3′-OH kinase (PI3K)/Akt/PKB pathway appearsimportant for regulating cell survival/cell death (Kulik et al. Mol.Cell. Biol. 17:1595-1606 (1997); Franke et al, Cell, 88:43-5437 (1997);Kauffmann-Zeh et al. Nature 385:544-548 (1997) Hemmings Science,275:628-630 (1997); Dudek et al., Science, 275:661-665 (1997)). Survivalfactors, such as platelet derived growth factor (PDGF), nerve growthfactor (NGF) and insulin-like growth factor-1 (IGF-1), promote cellsurvival under various conditions by inducing the activity of PI3K(Kulik et al. 1997, Hemmings 1997). Activated PI3K leads to theproduction of phosphatidylinositol (3,4,5)-triphosphate(PtdIns(3,4,5)-P3), which in turn binds to, and promotes the activationof, the serine/threonine kinase Akt, which contains a pleckstrinhomology (PH)-domain (Franke et al Cell, 81:727-736 (1995); HemmingsScience, 277:534 (1997); Downward, Curr. Opin. Cell Biol. 10:262-267(1998), Alessi et al., EMBO J. 15: 6541-6551 (1996)). Specificinhibitors of PI3K or dominant negative Akt/PKB mutants abolishsurvival-promoting activities of these growth factors or cytokines. Ithas been previously disclosed that inhibitors of PI3K (LY294002 orwortmannin) blocked the activation of Akt/PKB by upstream kinases. Inaddition, introduction of constitutively active PI3K or Akt/PKB mutantspromotes cell survival under conditions in which cells normally undergoapoptotic cell death (Kulik et al. 1997, Dudek et al. 1997).

Analysis of Akt levels in human tumors showed that Akt2 is overexpressedin a significant number of ovarian (J. Q. Cheung et al. Proc. Natl.Acad. Sci. U.S.A. 89:9267-9271 (1992)) and pancreatic cancers (J. Q.Cheung et al. Proc. Natl. Acad. Sci. U.S.A. 93:3636-3641 (1996)).Similarly, Akt3 was found to be overexpressed in breast and prostatecancer cell lines (Nakatani et al. J. Biol. Chem. 274:21528-21532(1999).

The tumor suppressor PTEN, a protein and lipid phosphatase thatspecifically removes the 3′ phosphate of PtdIns(3,4,5)-P3, is a negativeregulator of the PI3K/Akt pathway (Li et al. Science 275:1943-1947(1997), Stambolic et al. Cell 95:29-39 (1998), Sun et al. Proc. Natl.Acad. Sci. U.S.A. 96:6199-6204 (1999)). Germline mutations of PTEN areresponsible for human cancer syndromes such as Cowden disease (Liaw etal. Nature Genetics 16:64-67 (1997)). PTEN is deleted in a largepercentage of human tumors and tumor cell lines without functional PTENshow elevated levels of activated Akt (Li et al. supra, Guldberg et al.Cancer Research 57:3660-3663 (1997), Risinger et al. Cancer Research57:4736-4738 (1997)).

These observations demonstrate that the PI3K/Akt pathway plays importantroles for regulating cell survival or apoptosis in tumorigenesis.

Three members of the Akt/PKB subfamily of second-messenger regulatedserine/threonine protein kinases have been identified and termedAkt1/PKBα, Akt2/PKBβ, and Akt3/PKBγ respectively. The isoforms arehomologous, particularly in regions encoding the catalytic domains.Akt/PKBs are activated by phosphorylation events occurring in responseto PI3K signaling. PI3K phosphorylates membrane inositol phospholipids,generating the second messengers phosphatidylinositol3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate, whichhave been shown to bind to the PH domain of Akt/PKB. The current modelof Akt/PKB activation proposes recruitment of the enzyme to the membraneby 3′-phosphorylated phosphoinositides, where phosphorylation of theregulatory sites of Akt/PKB by the upstream kinases occurs (B. A.Hemmings, Science 275:628-630 (1997); B. A. Hemmings, Science 276:534(1997); J. Downward, Science 279:673-674 (1998)).

Phosphorylation of Akt1/PKBI occurs on two regulatory sites, Thr³⁰⁸ inthe catalytic domain activation loop and on Ser⁴⁷³ near the carboxyterminus (D. R. Alessi et al. EMBO J. 15:6541-6551 (1996) and R. Meieret al. J. Biol. Chem. 272:30491-30497 (1997)). Equivalent regulatoryphosphorylation sites occur in Akt2/PKBβ and Akt3/PKBγ. The upstreamkinase, which phosphorylates Akt/PKB at the activation loop site hasbeen cloned and termed 3′-phosphoinositide dependent protein kinase 1(PDK1). PDK1 phosphorylates not only Akt/PKB, but also p70 ribosomal S6kinase, p90RSK, serum and glucocorticoid-regulated kinase (SGK), andprotein kinase C. The upstream kinase phosphorylating the regulatorysite of Akt/PKB near the carboxy terminus has not been identified yet,but recent reports imply a role for the integrin-linked kinase (ILK-1),a serine/threonine protein kinase, or autophosphorylation.

Inhibition of Akt activation and activity can be achieved by inhibitingPI3K with inhibitors such as LY294002 and wortmannin. However, PI3Kinhibition has the potential to indiscriminately affect not just allthree Akt isozymes but also other PH domain-containing signalingmolecules that are dependent on PdtIns(3,4,5)-P3, such as the Tec familyof tyrosine kinases. Furthermore, it has been disclosed that Akt can beactivated by growth signals that are independent of PI3K.

Alternatively, Akt activity can be inhibited by blocking the activity ofthe upstream kinase PDK1. No specific PDK1 inhibitors have beendisclosed. Again, inhibition of PDK1 would result in inhibition ofmultiple protein kinases whose activities depend on PDK1, such asatypical PKC isoforms, SGK, and S6 kinases (Williams et al. Curr. Biol.10:439-448 (2000).

It is an object of the instant invention to provide novel compounds thatare inhibitors of Akt/PKB.

It is also an object of the present invention to provide pharmaceuticalcompositions that comprise.

It is also an object of the present invention to provide a method fortreating cancer that comprises administering such inhibitors of Akt/PKBactivity.

SUMMARY OF THE INVENTION

The instant invention provides for compounds that inhibit of Akt/PKBactivity. In particular, the compounds disclosed selectively inhibit oneor two of the Akt/PKB isoforms. The invention also provides forcompositions comprising such inhibitory compounds and methods ofinhibiting Akt/PKB activity by administering the compound to a patientin need of treatment of cancer.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of the instant invention are useful in the inhibition ofthe activity of the serine/threonine kinase Akt. In a first embodimentof this invention, the inhibitors of Akt activity are illustrated by theformula A:

wherein

-   -   R¹ represents phenyl, furyl, thienyl or pyridinyl, any of which        groups may be optionally substituted with one, two or three        substituents, independently selected from:    -   a) halogen;    -   b) C₁₋₄ alkyl;    -   c) C₁₋₄ alkoxy;    -   d) cyano;    -   e) di(C₁₋₄ alkyl)amino;    -   f) hydroxy;    -   R² represents amino-C₁₋₆ alkyl, C₁₋₄ alkylamino-(C₁₋₆)alkyl,        di(C₁₋₄ alkyl)amino-(C₁₋₆)alkyl, hydroxy-(C₁₋₆)alkyl or C₁₋₄        alkoxy-(C₁₋₆)alkyl, any of which groups may be optionally        substituted; and    -   R³ is selected from: C₃₋₇ cycloalkyl and aryl, any of which        groups may be optionally substituted;        or a pharmaceutically acceptable salt of stereoisomer thereof.

In another embodiment the inhibitors of the instant invention areillustrated by the formula A-I:

wherein

-   -   R² is as defined with reference to formula II above;    -   R³ is selected from: C₃₋₇ cycloalkyl and phenyl, any of which        groups may be optionally substituted.    -   m is 0, 1, 2 or 3; and    -   R⁵ independently represents halogen, C₁₋₄ alkyl or C₁₋₆ alkoxy;        or the pharmaceutically acceptable salts thereof.

Specific compounds of the instant invention include:

-   6-(2-hydroxyethyl)oxy-3,7-diphenyl-[1,2,4]triazolo[4,3-b]pyridazine-   6-(4-hydroxybutyl)oxy-3,7-diphenyl-[1,2,4]triazolo[4,3-b]pyridazine    or a pharmaceutically acceptable salt thereof.

As used herein, the expression “C₁₋₆ alkyl” includes methyl and ethylgroups, and straight-chained or branched propyl, butyl, pentyl and hexylgroups. Particular alkyl groups are methyl, ethyl, n-propyl, isopropyl,tert-butyl and 2,2-dimethylpropyl. Derived expressions such as “C₁₋₆alkoxy” are to be construed accordingly.

As used herein, the expression “C₁₋₄ alkyl” includes methyl and ethylgroups, and straight-chained or branched propyl and butyl groups.Particular alkyl groups are methyl, ethyl, n-propyl, isopropyl andtert-butyl. Derived expressions such as “C₁₋₄ alkoxy” are to beconstrued accordingly.

Typical C₃₋₇ cycloalkyl groups include cyclopropyl, cyclobutyl,cyclopentyl and cyclohexyl.

The expression “C₃₋₇ cycloalkyl(C₁₋₆)alkyl” as used herein includescyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl andcyclohexylmethyl.

Typical C₄₋₇ cycloalkenyl groups include cyclobutenyl, cyclopentenyl andcyclohexenyl.

Typical aryl groups include phenyl and naphthyl, preferably phenyl.

The expression “aryl(C₁₋₆)alkyl” as used herein includes benzyl,phenylethyl, phenylpropyl and naphthylmethyl.

The term “halogen” as used herein includes fluorine, chlorine, bromineand iodine, especially fluorine or chlorine.

For use in medicine, the salts of the compounds of formula A will bepharmaceutically acceptable salts. Other salts may, however, be usefulin the preparation of the compounds according to the invention or oftheir pharmaceutically acceptable salts. Suitable pharmaceuticallyacceptable salts of the compounds of this invention include acidaddition salts which may, for example, be formed by mixing a solution ofthe compound according to the invention with a solution of apharmaceutically acceptable acid such as hydrochloric acid, sulphuricacid, methanesulphonic acid, fumaric acid, maleic acid, succinic acid,acetic acid, benzoic acid, oxalic acid, citric acid, tartaric acid,carbonic acid or phosphoric acid. Furthermore, where the compounds ofthe invention carry an acidic moiety, suitable pharmaceuticallyacceptable salts thereof may include alkali metal salts, e.g. sodium orpotassium salts; alkaline earth metal salts, e.g. calcium or magnesiumsalts; and salts formed with suitable organic ligands, e.g. quaternaryammonium salts.

The present invention includes within its scope prodrugs of thecompounds of formula A above. In general, such prodrugs will befunctional derivatives of the compounds of formula A that are readilyconvertible in vivo into the required compound of formula A.Conventional procedures for the selection and preparation of suitableprodrug derivatives are described, for example, in Design of Prodrugs,ed. H. Bundgaard, Elsevier, 1985.

Where the compounds according to the invention have at least oneasymmetric center, they may accordingly exist as enantiomers. Where thecompounds according to the invention possess two or more asymmetriccenters, they may additionally exist as diastereoisomers. It is to beunderstood that all such isomers and mixtures thereof in any proportionare encompassed within the scope of the present invention.

Examples of suitable values for the substituent R³ includemethyl-cyclopropyl, cyclobutyl, methyl-cyclobutyl, cyclopentyl,methyl-cyclopentyl, cyclohexyl, cyclobutenyl and phenyl.

In a particular embodiment, the substituent R³ represents C₃₋₇cycloalkyl or phenyl, either unsubstituted or substituted by C₁₋₆ alkylor halogen, especially methyl or fluorine. Favourably, R³ representscyclobutyl or phenyl.

Representative values of R¹ include phenyl, methylphenyl, fluorophenyl,difluorophenyl and methoxyphenyl.

Examples of typical optional substituents on the group R¹ includemethyl, fluoro and methoxy.

In a particular embodiment, R² represents amino-C₁₋₆ alkyl, C₁₋₄alkylamino-(C₁₋₆)alkyl or di(C₁₋₄ alkyl)amino-(C₁₋₆)alkyl.Representative values of R² include but are not limited todimethylaminomethyl, aminoethyl, dimethylaminoethyl, diethylaminoethyl,3-dimethylaminopropyl, 3-methylaminopropyl,3-dimethylamino-2,2-dimethylpropyl and, 3-dimethylamino-2-methylpropyl.

Preferably, R³ represents hydrogen or methyl.

The compounds of the instant invention are inhibitors of the activity ofAkt and are thus useful in the treatment of cancer, in particularcancers associated with irregularities in the activity of Akt and/orGSK3. Such cancers include, but are not limited to ovarian, pancreaticand breast cancer.

In an embodiment of the invention, the instant compound is a selectiveinhibitor whose inhibitory efficacy is dependent on the PH domain. Inthis embodiment, the compound exhibits a decrease in in vitro inhibitoryactivity or no in vitro inhibitory activity against truncated Aktproteins lacking the PH domain.

In another embodiment of the invention, the instant compound is aselective inhibitor whose inhibitory efficacy is dependent on the regionof the proteins between the PH domain and the kinase domain. (SeeKonishi et al. Biochem. and Biophys. Res. Comm. 216: 526-534 (1995),FIG. 2) That region will be referred to as the hinge region. In thisembodiment, the compound exhibits a decrease in in vitro inhibitoryactivity or no in vitro inhibitory activity against truncated Aktproteins lacking the PH domain and the hinge region.

Such an inhibitor that is dependent on either the PH domain, the hingeregion or both provides a particular advantage since the PH domains andhinge regions in the three Akt isoforms lack the sequence homology thatis present in the rest of the protein, particularly the homology foundin the kinase domains (which comprise the catalytic domains andATP-binding consensus sequences). It is therefore observed that certaininhibitor compounds, such as those described herein, are not onlyselective for one or two isoforms of Akt, but also are weak inhibitorsor fail to inhibit other kinases, such as PKA and PKC, whose kinasedomains share some sequence homology with the kinase domains of theAkt/PKB isoforms. Both PKA and PKC lack a PH domain.

In a further embodiment, the instant compound is selected from the groupof a selective inhibitor of Akt 1, a selective inhibitor of Akt 2 and aselective inhibitor of both Akt 1 and Akt 2.

In another embodiment, the instant compound is selected from the groupof a selective inhibitor of Akt 1, a selective inhibitor of Akt 2, aselective inhibitor of Akt3 and a selective inhibitor of two of thethree Akt isoforms.

In another embodiment, the instant compound is a selective inhibitor ofall three Akt isoforms, but is not an inhibitor of one, two or all ofsuch Akt isoforms that have been modified to delete the PH domain, thehinge region or both the PH domain and the hinge region.

The present invention is further directed to a method of inhibiting Aktactivity which comprises administering to a mammal in need thereof apharmaceutically effective amount of the instant compound.

The compounds of this invention may be administered to mammals,preferably humans, either alone or, preferably, in combination withpharmaceutically acceptable carriers, excipients or diluents, in apharmaceutical composition, according to standard pharmaceuticalpractice. The compounds can be administered orally or parenterally,including the intravenous, intramuscular, intraperitoneal, subcutaneous,rectal and topical routes of administration.

The pharmaceutical compositions containing the active ingredient may bein a form suitable for oral use, for example, as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions, hard or soft capsules, or syrups or elixirs. Compositionsintended for oral use may be prepared according to any method known tothe art for the manufacture of pharmaceutical compositions and suchcompositions may contain one or more agents selected from the groupconsisting of sweetening agents, flavoring agents, coloring agents andpreserving agents in order to provide pharmaceutically elegant andpalatable preparations. Tablets contain the active ingredient inadmixture with non-toxic pharmaceutically acceptable excipients whichare suitable for the manufacture of tablets. These excipients may be forexample, inert diluents, such as calcium carbonate, sodium carbonate,lactose, calcium phosphate or sodium phosphate; granulating anddisintegrating agents, for example, microcrystalline cellulose, sodiumcrosscarmellose, corn starch, or alginic acid; binding agents, forexample starch, gelatin, polyvinyl-pyrrolidone or acacia, andlubricating agents, for example, magnesium stearate, stearic acid ortalc. The tablets may be uncoated or they may be coated by knowntechniques to mask the unpleasant taste of the drug or delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period. For example, a watersoluble taste masking material such as hydroxypropylmethyl-cellulose orhydroxypropylcellulose, or a time delay material such as ethylcellulose, cellulose acetate buryrate may be employed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with watersoluble carrier such as polyethyleneglycol or an oil medium, for examplepeanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active material in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose,sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethylene-oxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose, saccharin or aspartame.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of ananti-oxidant such as butylated hydroxyanisol or alpha-tocopherol.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present. These compositions may be preserved by theaddition of an anti-oxidant such as ascorbic acid.

The pharmaceutical compositions of the invention may also be in the formof an oil-in-water emulsions. The oily phase may be a vegetable oil, forexample olive oil or arachis oil, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifying agents may benaturally-occurring phosphatides, for example soy bean lecithin, andesters or partial esters derived from fatty acids and hexitolanhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening, flavouring agents, preservatives and antioxidants.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso contain a demulcent, a preservative, flavoring and coloring agentsand antioxidant.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous solutions. Among the acceptable vehicles and solventsthat may be employed are water, Ringer's solution and isotonic sodiumchloride solution.

The sterile injectable preparation may also be a sterile injectableoil-in-water microemulsion where the active ingredient is dissolved inthe oily phase. For example, the active ingredient may be firstdissolved in a mixture of soybean oil and lecithin. The oil solution isthen introduced into a water and glycerol mixture and processed to forma microemulation.

The injectable solutions or microemulsions may be introduced into apatient's blood-stream by local bolus injection. Alternatively, it maybe advantageous to administer the solution or microemulsion in such away as to maintain a constant circulating concentration of the instantcompound. In order to maintain such a constant concentration, acontinuous intravenous delivery device may be utilized. An example ofsuch a device is the Deltec CADD-PLUS™ model 5400 intravenous pump.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension for intramuscular andsubcutaneous administration. This suspension may be formulated accordingto the known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in 1,3-butane diol. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose any bland fixed oil may be employed includingsynthetic mono- or diglycerides. In addition, fatty acids such as oleicacid find use in the preparation of injectables.

Compounds of Formula A may also be administered in the form of asuppositories for rectal administration of the drug. These compositionscan be prepared by mixing the drug with a suitable non-irritatingexcipient which is solid at ordinary temperatures but liquid at therectal temperature and will therefore melt in the rectum to release thedrug. Such materials include cocoa butter, glycerinated gelatin,hydrogenated vegetable oils, mixtures of polyethylene glycols of variousmolecular weights and fatty acid esters of polyethylene glycol.

For topical use, creams, ointments, jellies, solutions or suspensions,etc., containing the compound of Formula A are employed. (For purposesof this application, topical application shall include mouth washes andgargles.)

The compounds for the present invention can be administered inintranasal form via topical use of suitable intranasal vehicles anddelivery devices, or via transdermal routes, using those forms oftransdermal skin patches well known to those of ordinary skill in theart. To be administered in the form of a transdermal delivery system,the dosage administration will, of course, be continuous rather thanintermittent throughout the dosage regimen.

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specific amounts, aswell as any product which results, directly or indirectly, fromcombination of the specific ingredients in the specified amounts.

The instant compounds may also be co-administered with other well knowntherapeutic agents that are selected for their particular usefulnessagainst the condition that is being treated. For example, the instantcompounds may be useful in combination with known anti-cancer andcytotoxic agents. Similarly, the instant compounds may be useful incombination with agents that are effective in the treatment andprevention of neurofibromatosis, restinosis, polycystic kidney disease,infections of hepatitis delta and related viruses and fungal infections.The instant compositions may also be useful in combination with otherinhibitors of parts of the signaling pathway that links cell surfacegrowth factor receptors to nuclear signals initiating cellularproliferation. Thus, the instant compounds may be utilized incombination with inhibitors of prenyl-protein transferase, includingprotein substrate competitive inhibitors of farnesyl-proteintransferase, farnesyl pyrophosphate competitive inhibitors of theactivity of farnesyl-protein transferase and/or inhibitors ofgeranylgeranyl-protein transferase. The instant compositions may also beco-administered with compounds that are selective inhibitors ofgeranylgeranyl protein transferase or selective inhibitors offarnesyl-protein transferase. The instant compositions may also beadministered in combination with a compound that has Raf antagonistactivity.

The compounds of the instant invention may also be co-administered withother well known cancer therapeutic agents that are selected for theirparticular usefulness against the condition that is being treated.Included in such combinations of therapeutic agents are combinationswith an antineoplastic agent. It is also understood that the instantcompositions and combinations may be used in conjunction with othermethods of treating cancer and/or tumors, including radiation therapyand surgery.

Examples of an antineoplastic agent include, in general,microtubule-stabilising agents (such as paclitaxel (also known asTaxol®), docetaxel (also known as Taxotere®), or their derivatives);alkylating agents, anti-metabolites; epidophyllotoxin; an antineoplasticenzyme; a topoisomerase inhibitor; procarbazine; mitoxantrone; platinumcoordination complexes; biological response modifiers and growthinhibitors; hormonal/anti-hormonal therapeutic agents and haematopoieticgrowth factors.

Example classes of antineoplastic agents include, for example, theanthracycline family of drugs, the vinca drugs, the mitomycins, thebleomycins, the cytotoxic nucleosides, the taxanes, the epothilones,discodermolide, the pteridine family of drugs, diynenes and thepodophyllotoxins. Particularly useful members of those classes include,for example, doxorubicin, carminomycin, daunorubicin, aminopterin,methotrexate, methopterin, dichloro-methotrexate, mitomycin C,porfiromycin, 5-fluorouracil, 6-mercaptopurine, gemcitabine, cytosinearabinoside, podophyllotoxin or podo-phyllotoxin derivatives such asetoposide, etoposide phosphate or teniposide, melphalan, vinblastine,vincristine, leurosidine, vindesine, leurosine, paclitaxel and the like.Other useful antineoplastic agents include estramustine, cisplatin,carboplatin, cyclophosphamide, bleomycin, gemcitibine, ifosarnide,melphalan, hexamethyl melamine, thiotepa, cytarabin, idatrexate,trimetrexate, dacarbazine, L-asparaginase, camptothecin, CPT-11,topotecan, ara-C, bicalutamide, flutamide, leuprolide, pyridobenzoindolederivatives, interferons and interleukins.

Additionally, compositions of the instant invention may also be usefulas radiation sensitizers. For instance, radiation therapy, includingx-rays or gamma rays that are delivered from either an externallyapplied beam or by implantation of tiny radioactive sources, may used incombination with the instant compounds to treat cancer.

If formulated as a fixed dose, such combination products employ thecombinations of this invention within the dosage range described belowand the other pharmaceutically active agent(s) within its approveddosage range. The compounds of the instant invention may alternativelybe used sequentially with known pharmaceutically acceptable agent(s)when a multiple combination formulation is inappropriate.

The instant compounds may also be useful in combination with an integrinantagonist for the treatment of cancer, as described in U.S. Ser. No.09/055,487, filed Apr. 6, 1998, which is incorporated herein byreference.

As used herein the term an integrin antagonist refers to compounds whichselectively antagonize, inhibit or counteract binding of a physiologicalligand to an integrin(s) that is involved in the regulation ofangiogenisis, or in the growth and invasiveness of tumor cells. Inparticular, the term refers to compounds which selectively antagonize,inhibit or counteract binding of a physiological ligand to the αvβ3integrin, which selectively antagonize, inhibit or counteract binding ofa physiological ligand to the αvβ5 integrin, which antagonize, inhibitor counteract binding of a physiological ligand to both the αvβ3integrin and the αvβ5 integrin, or which antagonize, inhibit orcounteract the activity of the particular integrin(s) expressed oncapillary endothelial cells. The term also refers to antagonists of theαvβ6, αvβ8, α1β1, α2β1, α5β1, α6β1 and α6β4 integrins. The term alsorefers antagonists of any combination of αvβ3, αvβ5, αvβ6, αvβ8, α1β1,α2β1, α5β1, α6β1 and α6β4 integrins. The instant compounds may also beuseful with other agents that inhibit angiogenisis and thereby inhibitthe growth and invasiveness of tumor cells, including, but not limitedto angiostatin and endostatin.

When a composition according to this invention is administered into ahuman subject, the daily dosage will normally be determined by theprescribing physician with the dosage generally varying according to theage, weight, and response of the individual patient, as well as theseverity of the patient's symptoms.

In one exemplary application, a suitable amount of the compound of theinstant invention is administered to a mammal undergoing treatment forcancer. Administration occurs in an amount of inhibitor of between about0.1 mg/kg of body weight to about 60 mg/kg of body weight per day,preferably of between 0.5 mg/kg of body weight to about 40 mg/kg of bodyweight per day. A particular therapeutic dosage that comprises theinstant composition includes from about 0.01 mg to about 1000 mg of theinstant compound. Preferably, the dosage comprises from about 1 mg toabout 1000 mg of the instant compound.

All patents, publications and pending patent applications identified arehereby incorporated by reference.

Abbreviations used in the description of the chemistry and in theExamples that follow are:

Ac₂O Acetic anhydride; Boc t-Butoxycarbonyl; DBU1,8-diazabicyclo[5.4.0]undec-7-ene; TFA: trifluoroacetic acid AA: aceticacid Boc/BOC t-Butoxycarbonyl; DMA dimethylacetamide DMFDimethylformamide; DMSO dimethyl sulfoxide; EDC1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride; EtOAcEthyl acetate; EtOH Ethanol; FAB Fast atom bombardment; HOAt1-Hydroxy-7-azabenzotriazole HOBt 1-Hydroxybenzotriazole hydrate; HOPO2-hydroxypyridine-N-oxide HPLC High-performance liquid chromatography;IPAc isopropylacetate MeOH methanol RPLC Reverse Phase LiquidChromatography THF Tetrahydrofuran.

Reactions used to generate the compounds of this invention are preparedby employing reactions as shown in the Schemes 1-2, in addition to otherstandard manipulations such as ester hydrolysis, cleavage of protectinggroups, etc., as may be known in the literature or exemplified in theexperimental procedures. Substituents R and R^(a), as shown in theSchemes, represent the substituents R¹ and R²; however their point ofattachment to the ring is illustrative only and is not meant to belimiting.

These reactions may be employed in a linear sequence to provide thecompounds of the invention or they may be used to synthesize fragmentswhich are subsequently joined by the alkylation reactions described inthe Schemes.

Synopsis of Schemes 1-2

The requisite intermediates are in some cases commercially available, orcan be prepared according to literature procedures. As illustrated inReaction Scheme 1, a suitably substituted phenylmaleic anyhydride I istreated with hydrazine to form the dihydropyridazone dione II.Subsequent oxidative chlorination and reaction with a suitablysubstituted benzoic hydrazide provide the 6-chlorotriazolo[4,3-b]pyridazine III. This intermediate can then be treatedwith a variety of alcohols to provide the instant compound IV.

Reaction Scheme 2 illustrates preparation of compounds of the instantinvention having a cycloalkyl substituent at the 7-position. While acyclobutyl group is illustrated, the sequence of reactions is generallyapplicable to incorporation of a variety of unsubstituted or substitutedcycloalkyl moieties. Thus, 3,6-dichloropyridazine is alkylated viasilver catalyzed oxidative decarboxylation with cyclobutyl carboxylicacid to provide the cyclobutyl dicloropyridazine V, which then undergoesthe reactions described above to provide the instant compound VI.

Reaction Scheme 3 illustrates an alternative preparation of the instantcompounds (Tetrahedron Letters 41:781-784 (2000).

EXAMPLES

Examples provided are intended to assist in a further understanding ofthe invention. Particular materials employed, species and conditions areintended to be further illustrative of the invention and not limitativeof the reasonable scope thereof.

Example 16-(2-Hydroxyethyl)oxy-3,7-diphenyl-[1,2,4]triazolo[4,3-b]pyridazine(Compound 1)

Step 1: 4-Phenyl-1,2-dihydropyridazine-3,6-dione

Phenylmaleic anhydride (30 g, 0.17 mol), sodium acetate trihydrate (28g, 0.21 mol) and hydrazine monohydrate (10 ml, 0.21 mol) were heatedtogether at reflux in 40% acetic acid (600 ml) for 18 h. The mixture wascooled at 7° C. for 2 h, then filtered. The solid was washed withdiethyl ether and dried in vacuo to give 11 g (34%) of the titlecompound: ¹H NMR (250 MHz, DMSO-d₆) δ 7.16 (1H, br s), 7.44 (5H, m),7.80 (2H, br s); MS (ES⁺) m/e 189 [MH⁺].

Step 2: 3,6 Dichloro-4-phenylpyridazine

4-Phenyl-1,2-dihydropyridazine-3,6-dione (3.4 g, 18 mmol) was heated atreflux in phosphorus oxychloride (70 ml) for 6 h. The solution wasconcentrated in vacuo, then the residue was dissolved in dichloromethane(100 ml) and was neutralised by the addition of cold 10% aqueous sodiumhydrogen carbonate (150 ml). The aqueous phase was washed withdichloromethane (2×50 ml), then the combined organic layers were washedwith saturated aqueous sodium chloride (50 ml), dried (Na₂SO₄), andconcentrated in vacuo to yield 3.9 g (97%) of the title compound: ¹H NMR(250 MHz, DMSO-d₆) δ 7.54-7.66 (5H, m) 8.14 (1H, s); MS (ES⁺) m/e225/227/229 [MH⁺].

Step 3: 6-Chloro-3,7-diphenyl-1,2,3-triazolo[4,3-b]pyridazine

3,6-Dichloro-4-phenylpyridazine (2.9 g, 13 mmol), benzoic hydrazide (1.9g, 21 mmol) and triethylammonium chloride (2.0 g, 14 mmol) were heatedtogether at reflux in xylene (150 ml) for three days. More benzoichydrazide (0.88 g, 6.5 mmol) was added and the mixture was heated asbefore for another day. The solvent was removed in vacuo, and theresidue was purified by flash chromatography (silica gel, 0-50%EtAOc/CH₂Cl₂) to afford 1.4 g (36%) of the title compound as a solid:

¹H NMR (250 MHz, CDCl₃) δ 7.55 (8H, m), 8.12 (1H, s), 8.50 (2H, m); MS(ES⁺) m/e 307/309 [MH⁺].

Step 4:6-(2-Hydroxyethyl)oxy-3,7-diphenyl-1,2,3-triazolo[4,3-b]pyridazine

Anhydrous DMF (1.5 ml) was added to a test tube containing NaH (13 mg)under nitrogen. Ethylene glycol (2 ml) was added and the mixture stirredat room temperature for 1 hour. The6-chloro-3,7-diphenyl-1,2,3-triazolo[4,3-b]pyridazine (50 mg (preparedas described in Step 3) was added as a solid and the reaction stirred atroom temperature for 30 mins. and then heated at 60 C for 8 hours andthen stirred 10 hours at room temperature. The reaction mixture was thenpoured over 20 ml of hot water, the mixture cooled and the aqueousmixture extracted with ether. The organic phases were combined, washedwith water, dried over MgSO₄, filtered and concentrated under vacuum toprovide the title compound.

¹H NMR (CDCl₃, 500 MHz at 20° C.) δ 8.48 (d, 2H, J=8.3), 8.04 (d, 1H,J=0.7), 7.61 (m, 2H), 7.57 (dd, 2H, J=7.6 and 8.1), 7.52 (m, 4H), 4.62(dd, 2H, J=3.9 and 5.1), 4.04 (d, 2H, J=3.7). LC/MS (ES+) [M+1]=333.2.

Example 26-(2-Hydroxybutyl)oxy-3,7-diphenyl-[1,2,4]triazolo[4,3-b]pyridazine(Compound 2)

The title compound was prepared by the procedure described in Example 1,but replacing ethylene glycol with 1,4-butanediol in Step 4.

¹H NMR (CDCl₃, 500 MHz at 20° C.) δ 8.52 (dd, 2H, J=7.8 and 1.5), 8.02(d, 1H, J=0.5), 7.58 (m, 4H), 7.51 (m, 4H), 4.53 (t, 2H, J=6.4), 3.69(app. t, 2H, J=5.5), 1.97 (m 2H), 1.72 (m, 2H). LC/MS (ES+) [M+1]=361.3.

Example 3

Cloning of the Human Akt Isoforms and ΔPH-Akt1

The pS2neo vector (deposited in the ATCC on Apr. 3, 2001 as ATCC) wasprepared as follows: The pRmHA3 vector (prepared as described in Nucl.Acid Res. 16:1043-1061 (1988)) was cut with BglII and a 2734 bp fragmentwas isolated. The pUChsneo vector (prepared as described in EMBO J.4:167-171 (1985)) was also cut with BglII and a 4029 bp band wasisolated. These two isolated fragments were ligated together to generatea vector termed pS2neo-1. This plasmid contains a polylinker between ametallothionine promoter and an alcohol dehydrogenase poly A additionsite. It also has a neo resistance gene driven by a heat shock promoter.The pS2neo-1 vector was cut with Psp5II and BsiWI. Two complementaryoligonucleotides were synthesized and then annealed (CTGCGGCCGC(SEQ.ID.NO.: 1) and GTACGCGGCCGCAG (SEQ.ID.NO.: 2)). The cut pS2neo-1and the annealed oligonucleotides were ligated together to generate asecond vector, pS2neo. Added in this conversion was a NotI site to aidin the linearization prior to transfection into S2 cells.

Human Akt1 gene was amplified by PCR (Clontech) out of a human spleencDNA (Clontech) using the 5′ primer: 5′CGCGAATTCAGATCTACCASTEAGCGACGTGGCTATTGTG 3′ (SEQ.ID.NO.: 3), and the 3′primer: 5′ CGCTCTAGAGGATCCTCAGGCCGTGCTGCTGGC3′(SEQ.ID.NO.: 4). The 5′primer included an EcoRI and BglII site. The 3′ primer included an XbaIand BamHI site for cloning purposes. The resultant PCR product wassubcloned into pGEM3Z (Promega) as an EcoRI/Xba I fragment. Forexpression/purification purposes, a middle T tag was added to the 5′ endof the full length Akt1 gene using the PCR primer:5′GTACGATGCTGAACGATATCTTCG 3′ (SEQ.ID.NO.: 5). The resulting PCR productencompassed a 5′ KpnI site and a 3′ BamHI site which were used tosubclone the fragment in frame with a biotin tag containing insect cellexpression vector, pS2neo.

For the expression of a pleckstrin homology domain (PH) deleted (Δ aa4-129, which includes deletion of a portion of the Akt1 hinge region)version of Akt1, PCR deletion mutagenesis was done using the full lengthAkt1 gene in the pS2neo vector as template. The PCR was carried out in 2steps using overlapping internal primers(5′GAATACATGCCGATGGAAAGCGACΔGGGGCTGAAGAGATGGAGGTG 3′ (SEQ.ID.NO.: 6),and 5′CCCCTCCATCTCTTCAGCCCCΔGTCGCTTTCCATCGGCATG TATTC 3′ (SEQ.ID.NO.:7)) which encompassed the deletion and 5′ and 3′ flanking primers whichencompassed the KpnI site and middle T tag on the 5′ end. The final PCRproduct was digested with KpnI and SmaI and ligated into the pS2neo fulllength Akt1 KpnI/Sma I cut vector, effectively replacing the 5′ end ofthe clone with the deleted version.

Human Akt3 gene was amplified by PCR of adult brain cDNA (Clontech)using the amino terminal oligo primer: 5′GAATTCAGATCTACCATGAGCGATGTTACCATTGTG 3′ (SEQ.ID.NO.: 8); and the carboxyterminal oligo primer: 5′TCTAGATCTTATTCTCGTCCACTTGCAGAG 3′ (SEQ.ID.NO.:9). These primers included a 5′ EcoRI/BglII site and a 3′ XbaI/BglIIsite for cloning purposes. The resultant PCR product was cloned into theEcoRI and XbaI sites of pGEM4Z (Promega). For expression/purificationpurposes, a middle T tag was added to the 5′ end of the full length Akt3clone using the PCR primer: 5′GGTACCATGGAATACATGCCGATGGAAAGCGATGTTACCATTGTGAAG 3′(SEQ.ID.NO.: 10). Theresultant PCR product encompassed a 5′ KpnI site which allowed in framecloning with the biotin tag containing insect cell expression vector,pS2neo.

Human Akt2 gene was amplified by PCR from human thymus cDNA (Clontech)using the amino terminal oligo primer: 5′AAGCTTAGATCTACCATGAATGAGGTGTCTGTC 3′ (SEQ.ID.NO.: 11); and the carboxyterminal oligo primer: 5′ GAATTCGGATCCTCACTCGCGGATGCTGGC 3′ (SEQ.ID.NO.:12). These primers included a 5′ HindIII/BglII site and a 3′ EcoRI/BamHIsite for cloning purposes. The resultant PCR product was subcloned intothe HindIII/EcoRI sites of pGem3Z (Promega). For expression/purificationpurposes, a middle T tag was added to the 5′ end of the full length Akt2using the PCR primer: 5′GGTACCATGGAATACATGCCGATGGAAAATGAGGTGTCTGTCATCAAAG 3′ (SEQ.ID.NO.: 13).The resultant PCR product was subcloned into the pS2neo vector asdescribed above.

Example 4

Expression of Human Akt Isoforms and ΔPH-Akt1

The DNA containing the cloned Akt1, Akt2, Akt3 and ΔPH-Akt1 genes in thepS2neo expression vector was purified and used to transfect DrosophilaS2 cells (ATCC) by the calcium phosphate method. Pools of antibiotic(G418, 500 μg/ml) resistant cells were selected. Cell were expanded to a1.0L volume (˜7.0×10⁶/ml), biotin and CuSO₄ were added to a finalconcentration of 50 μM and 50 mM respectively. Cells were grown for 72hat 27° C. and harvested by centrifugation. The cell paste was frozen at−70° C. until needed.

Example 5

Purification of Human Akt Isoforms and ΔPH-Akt1

Cell paste from one liter of S2 cells, described in Example 13, waslysed by sonication with 50 mls 1% CHAPS in buffer A: (50 mM Tris pH7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM AEBSF, 10 μg/ml benzamidine, 5 μg/mlof leupeptin, aprotinin and pepstatin each, 10% glycerol and 1 mM DTT).The soluble fraction was purified on a Protein G Sepharose fast flow(Pharmacia) column loaded with 9 mg/ml anti-middle T monoclonal antibodyand eluted with 75 μM EYMPME (SEQ.ID.NO.: 14) peptide in buffer Acontaining 25% glycerol. Akt/PKB containing fractions were pooled andthe protein purity evaluated by SDS-PAGE. The purified protein wasquantitated using a standard Bradford protocol. Purified protein wasflash frozen on liquid nitrogen and stored at −70° C.

Example 6

Kinase Assays

This procedure describes a kinase assay which measures phosphorylationof a biotinylated GSK3-derived peptide by human recombinant activeAkt/PBK isoforms or Akt/PBK mutants. The ³³P-labeled biotinylatedproduct can be captured and detected using Streptavidin coatedFlashplates (NEN LifeSciences) or Streptavidin Membrane Filter Plates(Promega). Alternatively, a GSK3-derived peptide with 2 added lysineresidues was used as the substrate and subsequently captured usingPhosphocellulose Membrane Filter Plates (Polyfiltronics).

Materials:

Active human Akt: The following active human Akt isoforms were utilizedin the in vitro assays: active human Akt1 (obtained from UpstateBiotechnology, catalog no. 14-276, 15 μg/37 μl (6.76 μM)) or recombinantlipid activated Akt1 (prepared as described in Example 5); Akt2(prepared as described in Example 5); Akt3 (prepared as described inExample 5); and delta PH-Akt1 (prepared as described in Example 5).

Akt specific peptide substrate: GSK3α (S21) Peptide #3928,biotin-GGRARTSSFAEPG (SEQ.ID.NO.: 15), FW=1517.8 (obtained fromMacromolecular Resources) for Streptavidin Flashplate or StreptavidinFilter Plate detection.

GSK3α (S21) Peptide #G80613, KKGGRARTSSFAEPG (SEQ.ID.NO.: 14), FW=1547.8(obtained from Research Genetics) for Phosphocellulose filter platedetection.

Standard Assay Solutions:

-   A.

10X Assay Buffer: 500 mM HEPES, pH 7.5 1% PEG  1 mM EDTA  1 mM EGTA  20mM β-Glycerol phosphate

-   B. Active Akt (500 nM): Diluent (1× Assay buffer, 10% glycerol, 0.1%    β-mercaptoethanol, 1.0 μM microcystin LR and 1.0 mM EDTA) was added    to a vial containing 37 μl of active Akt isoform (6.76 μM). Aliquots    were flash frozen in liquid N₂ and stored at −70° C.-   C. 1 mM Akt specific peptide substrate in 50 mM Tris pH 7.5, 1 mM    DTT.-   D. 100 mM DTT in di H₂O.-   E. 100× Protease Inhibitor Cocktail (PIC): 1 mg/ml benzamidine, 0.5    mg/ml pepstatin, 0.5 mg/ml leupeptin, 0.5 mg/ml aprotinin.-   F. 3 mM ATP, 200 mM MgCl₂ in H₂O, pH 7.9.-   G. 50% (v/v) Glycerol.-   H. 1% (wt/v) BSA (10 mg/ml) in diH20, 0.02% (w/v) NaN₃.-   I. 125 mM EDTA.-   J. 0.75% (wt/v) Phosphoric Acid.-   K. 2.5 M Potassium Chloride.-   L. Tris Buffered Saline (TBS), 25 mM Tris, 0.15 M Sodium Chloride,    pH 7.2 (BupH Tris Buffered Saline Pack, Pierce catalog no. 28376).    Procedure for Streptavidin Flash Plate Assay:    Step 1:

A 1 μl solution of the test compound in 100% DMSO was added to 20 μl of2× substrate solution (20 uM GSK3 Peptide, 300 TM ATP, 20 mM MgCl₂, 20μCi/ml [γ³³P] ATP, 1× Assay Buffer, 5% glycerol, 1 mM DTT, 1×PIC, 0.1%BSA and 100 mM KCl). Phosphorylation reactions were initiated by adding19 μl of 2× Enzyme solution (6.4 nM active Akt/PKB, 1× Assay Buffer, 5%glycerol, 1 mM DTT, 1×PIC and 0.1% BSA). The reactions were thenincubated at room temperature for 45 minutes.

Step 2:

The reaction was stopped by adding 170 μl of 125 mM EDTA. 200 μl ofstopped reaction was transferred to a Streptavidin Flashplate® PLUS (NENLife Sciences, catalog no. SMP103). The plate was incubated for ≧10minutes at room temperature on a plate shaker. The contents of each wellwas aspirated, and the wells rinsed 2 times with 200 μl TBS per well.The wells were then washed 3 times for 5 minutes with 200 μl TBS perwell with the plates incubated at room temperature on a platform shakerduring wash steps.

The plates were covered with sealing tape and counted using the PackardTopCount with the appropriate settings for counting [³³P] inFlashplates.

Procedure for Streptavidin Filter Plate Assay:

Step 1:

The enzymatic reactions as described in Step 1 of the Streptavidin FlashPlate Assay above were performed.

Step 2:

The reaction was stopped by adding 20 μl of 7.5M GuanidineHydrochloride. 50 μl of the stopped reaction was transferred to theStreptavidin filter plate (SAM²™ Biotin Capture Plate, Promega, catalogno. V7542) and the reaction was incubated on the filter for 1-2 minutesbefore applying vacuum.

The plate was then washed using a vacuum manifold as follows: 1) 4×200μl/well of 2M NaCl; 2) 6×200 μl/well of 2M NaCl with 1% H₃PO₄; 3) 2×200μl/well diH₂O; and 4) 2×100 μl/well of 95% Ethanol. The membranes werethen allowed to air dry completely before adding scintillant.

The bottom of the plate was sealed with white backing tape, 30 μl/wellof Microscint 20 (Packard Instruments, catalog no. 6013621) was added.The top of the plate was sealed with clear sealing tape, and the platethen counted using the Packard TopCount with the appropriate settingsfor [³³P] with liquid scintillant.

Procedure for Phosphocellulose Filter Plate Assay:

Step 1:

The enzymatic reactions were performed as described in Step 1 of theStreptavidin Flash Plate Assay (above) utilizing KKGGRARTSSFAEPG(SEQ.ID.NO.: 16) as the substrate in place of biotin-GGRARTSSFAEPG.

Step 2:

The reaction was stopped by adding 20 μl of 0.75% H₃PO₄. 50 μl ofstopped reaction was transferred to the filter plate (UNIFILTER™,Whatman P81 Strong Cation Exchanger, White Polystyrene 96 Well Plates,Polyfiltronics, catalog no. 7700-3312) and the reaction incubated on thefilter for 1-2 minutes before applying vacuum.

The plate was then washed using a vacuum manifold as follows: 1) 9×200μl/well of 0.75% H₃PO₄; and 2) 2×200 μl/well of diH₂O. The bottom of theplate was sealed with white backing tape, then 30 μl/well of Microscint20 was added. The top of the plate was sealed with clear sealing tape,and the plate counted using the Packard TopCount with the appropriatesettings for [³³P] and liquid scintillant.

PKA Assay

Each individual PKA assay consists of the following components:

-   1) 10 μl 5×PKA assay buffer (200 mM Tris pH7.5, 100 mM MgCl₂, 5 mM    2-mercaptoethanol, 0.5 mM EDTA)-   2) 10 μl of a 50 μM stock of Kemptide (Sigma) diluted into water-   3) 10 μl ³³P-ATP (prepared by diluting 1.0 μl ³³P-ATP [10 mCi/ml]    into 200 μl of a 50 μM stock of unlabeled ATP)-   4) 10 μl appropriate solvent control dilution or inhibitor dilution-   5) 10 μl of a 70 nM stock of PKA catalytic subunit (UBI catalog    #14-114) diluted in 0.5 mg/ml BSA

The final assay concentrations were 40 mM Tris pH 7.5, 20 mM MgCl₂, 1 mM2-mercaptoethanol, 0.1 mM EDTA, 10 μM Kemptide, 10 μM ³³P-ATP, 14 nM PKAand 0.1 mg/ml BSA.

Assays were assembled in 96 deep-well assay plates. Components #3 and #4were premixed and in a separate tube, a mixture containing equal volumesof components #1, #2, and #5 was prepared. The assay reaction wasinitiated by adding 30 μl of the components #1, #2, and #5 mixture towells containing ³³P-ATP and inhibitor. The liquid in the assay wellswas mixed and the assay reactions incubated for 20 minutes at roomtemperature. The reactions were stopped by adding 50 μl 100 mM EDTA and100 mM sodium pyrophosphate and mixing.

The enzyme reaction product (phosphorylated Kemptide) was quantitatedusing p81 phosphocellulose 96 well filter plates (Millipore). Each wellof a p81 filter plate was filled with 75 mM phosphoric acid. The wellswere aspirated and 170 μl of 75 mM phosphoric acid was added to eachwell. A 30-40 μl aliquot from each stopped PKA reaction was added tocorresponding wells on the filter plate containing the phosphoric acid.The peptide was trapped on the filter following the application of avacuum. The filters were washed 5× by filling wells with 75 mMphosphoric acid followed by aspiration. After the final wash, thefilters were allowed to air dry. 30 μl scintillation fluid was added toeach well and the filters counted on a TopCount (Packard).

PKC Assay

Each PKC assay consists of the following components:

-   1) 5 μl 10×PKC co-activation buffer (2.5 mM EGTA, 4 mM CaCl₂)-   2) 10 μl 5×PKC activation buffer (1.6 mg/ml phosphatidylserine, 0.16    mg/ml diacylglycerol, 100 mM Tris pH 7.5, 50 mM MgCl, 5 mM    2-mercaptoethanol)-   3) 5 μl ³³P-ATP (prepared by diluting 1.0 μl ³³P-ATP [10 mCi/ml]    into 100 μl of a 100 μM stock of unlabeled ATP)-   4) 10 μl of a 350 μg/ml stock of myelin basic protein (MBP, UBI)    diluted in water-   5) 10 μl appropriate solvent control or inhibitor dilution-   6) 10 μl of a 50 ng/ml stock of PKC (mix of isoforms from UBI    catalog #14-115) diluted into 0.5 mg/ml BSA

Final assay concentrations were as follows: 0.25 mM EGTA, 0.4 mM CaCl,20 mM Tris pH 7.5, 10 mM MgCl, 1 mM 2-mercaptoethanol, 0.32 mg/mlphosphatidylserine, 0.032 mg/ml diacylglycerol, 10 μM ³³P-ATP, 70 μg/mlMBP, 10 ng/ml PKC, 0.1 mg/ml BSA.

Assays are performed using 96 deep well assay plates. In each assay well10 μl of solvent control or appropriate inhibitor dilution with 5 μl³³P-ATP (components #5 and #3) were premixed. In a separate tube, amixture containing equal volumes of components #1, #2, #4, and #6 wasprepared. The assay reaction was initiated by adding 35 μl of thecomponents #1, #2, #4, and #6 mixture to wells containing ³³P-ATP andinhibitor. The liquid in the assay wells was thoroughly mixed and theassay reactions incubated for 20 minutes at room temperature. Thereactions were stopped by adding 100 mM EDTA (50 μl) and 100 mM sodiumpyrophosphate (50 μl) and mixing. Phosphorylated MBP was collected onPVDF membranes in 96 well filter plates and quantitated by scintillationcounting.

The results from testing the compounds described in Examples 1-2 in theassays described above are shown in Table 1:

TABLE 1 GSK3 Peptide Substrate Counter IC₅₀ (μM) screens Akt-1 deltaIC₅₀ (μM) Akt-1 PH Akt2 Akt3 PKA PKC Compound 1 15.9 >50 >50 >50 >40 >40Compound 2 4.65 >50 >50 >50 >40 >40

Example 7

Cell Based Assays to Determine Inhibition of Akt/PKB

Cells (for example LnCaP or a PTEN^((−/−)) tumor cell line withactivated Akt/PKB) were plated in 100 mM dishes. When the cells wereapproximately 70 to 80% confluent, the cells were refed with 5 mls offresh media and the test compound added in solution. Controls includeduntreated cells, vehicle treated cells and cells treated with eitherLY294002 (Sigma) or wortmanin (Sigma) at 20 μM or 200 nM, respectively.The cells were incubated for 2 hrs, and the media removed, The cellswere washed with PBS, scraped and transferred to a centrifuge tube. Theywere pelleted and washed again with PBS. Finally, the cell pellet wasresuspended in lysis buffer (20 mM Tris pH8, 140 mM NaCl, 2 mM EDTA, 1%Triton, 1 mM Na Pyrophosphate, 10 mM β-Glycerol Phosphate, 10 mM NaF,0.5 mm NaVO₄, 1 μM Microsystine, and 1× Protease Inhibitor Cocktail),placed on ice for 15 minutes and gently vortexed to lyse the cells. Thelysate was spun in a Beckman tabletop ultra centrifuge at 100,000×g at4° C. for 20 min. The supernatant protein was quantitated by a standardBradford protocol (BioRad) and stored at −70° C. until needed.

Proteins were immunoprecipitated (IP) from cleared lysates as follows:For Akt1/PKBα, lysates are mixed with Santa Cruz sc-7126 (D-17) in NETN(100 mM NaCl, 20 mM Tris pH 8.0, 1 mM EDTA, 0.5% NP40) and Protein A/GAgarose (Santa Cruz sc-2003) was added. For Akt2/PKBβ, lysates weremixed in NETN with anti-Akt-2 agarose (Upstate Biotechnology #16-174)and for Akt3/PKBγ, lysates were mixed in NETN with anti-Akt-3 agarose(Upstate Biotechnology #16-175). The IPs were incubated overnight at 4°C., washed and seperated by SDS-PAGE.

Western blots were used to analyze total Akt, pThr308 Akt, pSer473 Akt,and downstream targets of Akt using specific antibodies (Cell SignalingTechnology): Anti-Total Akt (cat. no. 9272), Anti-Phopho Akt Serine 473(cat. no. 9271), and Anti-Phospho Akt Threonine 308 (cat. no. 9275).After incubating with the appropriate primary antibody diluted inPBS+0.5% non-fat dry milk (NFDM) at 4° C. overnight, blots were washed,incubated with Horseradish peroxidase (HRP)-tagged secondary antibody inPBS+0.5% NFDM for 1 hour at room temperature. Proteins were detectedwith ECL Reagents (Amersham/Pharmacia Biotech RPN2134).

Example 8

Heregulin Stimulated Akt Activation

MCF7 cells (a human breast cancer line that is PTEN^(+/+)) were platedat 1×10⁶ cells per 100 mM plate. When the cells were 70-80% confluent,they were refed with 5 ml of serum free media and incubated overnight.The following morning, compound was added and the cells were incubatedfor 1-2 hrs, heregulin was added (to induce the activation of Akt) for30 minutes and the cells were analyzed as described above.

Example 9

Inhibition of Tumor Growth

In vivo efficacy of an inhibitor of the growth of cancer cells may beconfirmed by several protocols well known in the art.

Human tumor cell lines which exhibit a deregulation of the PI3K pathway(such as LnCaP, PC3, C33a, OVCAR-3, MDA-MB-468 or the like) are injectedsubcutaneously into the left flank of 8-12 week old female nude mice(Harlan) on day 0. The mice are randomly assigned to a vehicle, compoundor combination treatment group. Daily subcutaneous administration beginson day 1 and continues for the duration of the experiment.Alternatively, the inhibitor test compound may be administered by acontinuous infusion pump. Compound, compound combination or vehicle isdelivered in a total volume of 0.1 ml. Tumors are excised and weighedwhen all of the vehicle-treated animals exhibited lesions of 0.5-1.0 cmin diameter, typically 4 to 5.5 weeks after the cells were injected. Theaverage weight of the tumors in each treatment group for each cell lineis calculated.

1. A compound of the formula A:

wherein R¹ represents phenyl, furyl, thienyl or pyridinyl, any of whichgroups may be optionally substituted with one, two or threesubstituents, independently selected from: a) halogen; b) C₁₋₄ alkyl; c)C₁₋₄ alkoxy; d) cyano; e) di(C₁₋₄ alkyl)amino; f) hydroxy; R² representsamino-C₁₋₆ alkyl, C₁₋₄ alkylamino-(C₁₋₆)alkyl, di(C₁₋₄alkyl)amino-(C₁₋₆)alkyl, or C₁₋₄ alkoxy-(C₁₋₆)alkyl; and R³ is selectedfrom: C₃₋₇ cycloalkyl and aryl; or a pharmaceutically acceptable salt orstereoisomer thereof.
 2. The compound according to claim 1 of theformula A-I:

wherein R² is as defined in claim 1; R³ is selected from: C₃₋₇cycloalkyl and phenyl, m is 0, 1, 2 or 3; and R⁵ independentlyrepresents halogen, C₁₋₄ alkyl or C₁₋₆ alkoxy; or a pharmaceuticallyacceptable salt or stereoisomer thereof.
 3. A pharmaceutical compositioncomprising a pharmaceutical carrier, and dispersed therein, atherapeutically effective amount of a compound of claim
 1. 4. A methodfor treating ovarian, pancreatic and breast cancer which comprisesadministering to a mammal in need thereof a therapeutically effectiveamount of a composition of claim
 3. 5. A method for treating ovarian,pancreatic and breast cancer which comprises administering to a mammalin need thereof a therapeutically effective amount of a compoundselected from6-(2-hydroxyethyl)oxy-3,7-diphenyl-[1,2,4]triazolo[4,3-b]pyridazine,6-(4-hydroxybutyl)oxy-3,7-diphenyl-[1,2,4]triazolo[4,3-b]pyridazine or apharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier.
 6. A pharmaceutical composition made by combiningthe compound of claim 1 and a pharmaceutically acceptable carrier.
 7. Aprocess for making a pharmaceutical composition comprising combining acompound of claim 1 and a pharmaceutically acceptable carrier.